Recent evidence increasingly supports the active role of integrins in lymphoid cell activation and proliferation. Our studies of gamma/delta T cells, expressing the B3 integrin vitronectin receptor (VNR), reflect some of the consequences this active interaction between lymphocytes and the extracellular matrix (ECM) proteins could have for T cell activation and differentiation. The VNR has been described as a T cell costimulatory molecule. We recently reported that the VNR has the potential to stimulate cytokine secretion in T cell hybridomas without involvement of T cell receptor-mediated signal i.e. without antigen recognition. Further studies demonstrated tyrosine phosphorylation of proteins following VNR cross-linking and the interaction of the VNR with protein kinases. This knowledge is important to better understand the role of adhesion molecules, the ECM, and the cellular microenvironment for lymphocyte activation and differentiation. A class of adhesion molecules, the VLA integrins, are expressed on thymocytes and have been shown to affect immature thymocyte differentiation in vitro. This study examined the ability of cAMP to regulate VLA receptor function in thymocytes. Pharmacologic agents that raise intracellular cAMP enhanced the binding of immature CD4- CD8- and CD4+ CD8+ thymocytes to fibronectin while having no effect on the binding of the more mature J11d- thymocytes. PGE2, a hormone produced by thymic epithelial cells and known to raise intracellular cAMP levels in thymocytes, also increased the binding of immature thymocytes to fibronectin. In contrast, activation of protein kinase C via PMA enhanced the binding of all three thymocyte subsets. The cAMP-induced binding was blocked by MAbs to the VLA integrin chains alpha4 and alpha5 by the protein kinase A (PKA) inhibitor, (Rp)-cAMPS, indicating that activation of PKA enhances VLA-4 and VLA-5 receptor function. Activation of PKA was induced in all three thymocyte subsets following addition of cAMPa or forskolin, indicating that the inability of cAMP to enhance the binding of J11d- thymocytes was not due to an inability to activate PKA. Thus, cAMP enhances integrin function in thymocytes in a maturation stage-specific manner.